ABSTRACT: Background. Multiple Sclerosis is a chronic inflammatory disease of the central nervous system (CNS) characterized by autoimmune demyelination and subsequent neuro-axonal degeneration. Despite major progress in deciphering MS immunopathogenesis and treating early stages of disease, CNS-confined processes underpinning later progressive form of MS remain elusive, alluding to the poor accessibility to the target organ. We and others have demonstrated the reliability of profiling DNA methylation, a stable epigenetic marker of genome activity, in neuronal nuclei or bulk tissue to capture relevant molecular changes underlying MS neuropathology. In this study, we examined DNA methylation changes occurring in the normal appearing white matter (NAWM) glial cells of MS patients in comparison to white matter (WM) of non-neurological controls (NNC). Results. We profiled DNA methylation in NeuN-negative nuclei of glial cells, isolated from 38 postmortem NAWM specimens of MS patients (n=8) in comparison to WM of NNC individuals (n=14), using Infinium MethylationEPIC BeadChip. We applied reference-free cell type deconvolution to further reduce cellular heterogeneity and identified 1,226 significant (genome-wide adjusted P-value < 0.05) differentially methylated positions (DMPs) between MS patients and controls. Functional annotation of the affected genes uncovered alterations of processes related to cellular motility, cytoskeleton dynamics, metabolic processes, synaptic support, neuroinflammation as well as signaling pathways, such as Rho, Wnt and TGF-β signaling. A fraction of these changes affected genes which displayed transcriptional differences in the brain of MS patients, as reported by publically available transcriptomic data. Gene ontology analysis of differentially methylated genes according to their constitutive cell type-specific expression in glial cells attributed alteration of cytoskeleton rearrangement and extracellular matrix remodelling to all glial cell types, while some processes, including ion transport, Wnt/TGF-β signaling and immune processes were more specifically linked to oligodendrocytes, astrocytes and microglial cells, respectively. Conclusion. Our findings strongly suggest that NAWM glial cells are highly active, even in the absence of lesional insult, collectively exhibiting a multicellular reaction in response to diffuse inflammation.
